The in-house 3′ bulk RNA-sequencing “3B-seq” method was modified from the single cell Drop-seq protocol36, as described37. Briefly, 10 ng of RNA was mixed with indexing oligonucleotides (Integrated DNA Technologies, Coralville, IA) and after 5 min of incubation, the RNA was combined with RT mix (1× Maxima RT buffer, 1 mmol/L deoxynucleoside triphosphate, 10 U/μL Maxima H-RTase (all Thermo Fisher Scientific), 1 U/μL RNase inhibitor (Lucigen, Middleton, WI), and 2.5 μmol/L Template Switch Oligo (TSO; Integrated DNA Technologies, Coralville, IA)). Primers are listed in Supplementary Table 4. Samples were incubated in a T100 thermal cycler (Bio-Rad) for 30 min at 22 °C and 90 min at 52 °C. The constructed complementary DNA (cDNA) was amplified by PCR using the RT mix as template and adding 1× HiFi HotStart Readymix (Kapa Biosystems, Wilmington, MA), and 0.8 μmol/L SMART PCR primer. The samples were thermocycled in a T100 thermocycler (Bio-Rad) as follows: 95 °C for 3 min; then 4 cycles of 98 °C for 20 s, 65 °C for 45 s, 72 °C for 3 min; then 16 cycles of 98 °C for 20 s, 67 °C for 20 s, 72 °C for 3 min; and with the final extension step of 5 min at 72 °C. An aliquot of this PCR product was used for TCR sequencing (below). The PCR products were pooled together in sets of 12 samples containing different indexing oligos and purified with 0.6× HighPrep PCR reagent (MAGBIO, Gaithersburg, USA) according to the manufacturer’s instructions. They were eluted in 10 μL of molecular grade water. The 3′-end complementary DNA fragments for sequencing were prepared using the Nextera XT (Illumina) tagmentation reaction. The reaction was performed according to the manufacturer’s instructions, except for the use of P5 SMART primer, which replaced the S5xx Nextera primer. Each set of 12 samples that was pooled after the PCR reaction was tagmented with a different Nextera N7xx index. Subsequently, the samples were PCR amplified as follows: 95 °C for 30 seconds, 11 cycles of 95 °C for 10 seconds, 55 °C for 30 seconds, and 72 °C for 30 seconds, with the final extension step of 5 minutes at 72 °C. Samples were purified twice using 0.6× and 1.0× HighPrep PCR reagent and eluted in 10 μL of molecular-grade water. The concentration of the libraries was measured by using a Qubit 2 fluorometer (Invitrogen) and the Qubit DNA HS Assay Kit (Thermo Fisher Scientific). The quality of the sequencing libraries was assessed using the LabChip GXII Touch HT electrophoresis system (PerkinElmer), with the DNA High Sensitivity Assay (PerkinElmer) and the DNA 5K/RNA/Charge Variant Assay LabChip (PerkinElmer). Samples were stored at −20 °C. The libraries were sequenced on an Illumina NextSeq 500 instrument, with a custom primer (DS Custom Read 1) producing read 1 of 20 base pairs (bp), and read 2 (paired end) of 50 bp. Sequencing was performed at the Functional Genomics Unit of the University of Helsinki, Finland.

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